Conference Abstract: Itch e3 ubiquitin ligase influences downstream receptor tyrosine kinase signalling in the oe33 cell line

Aidan Seeley, Neville Cobbe, Richard Turkington, Daniel Longley, Emma Evergren

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Introduction: Deregulation of the receptor tyrosine kinases EGFR and Met is an important driver of cancer cell development and progression to metastasis. Trafficking regulates ERK1/2, STAT3, Gab1 and Rac1 signalling pathways; that have been implicated in cell survival, invasion and metastasis. Cell migration, metastasis and tumorigenesis have been shown to be promoted where mutations affect endocytic trafficking due to increased endosomal signalling. Advancements in our understanding of regulation of EGFR and Met signalling are essential to understand their role in cancer progression. Receptor degradation and membrane trafficking are both regulated by ubiquitination. Itch, an E3 ubiquitin ligase, has been shown to increase tumourgenicity in breast cancer and affect EGFR signalling. ​
Hypothesis: Here we investigated the role of the ubiquitin E3 ligase Itch in endocytosis and EGFR and Met downstream signalling in the oesophageal cancer cell line OE33.​
Method: Stable Itch knockdown and overexpression cell lines were generated by transfection of oesophageal adenocarcinoma OE33 cells with lentivirus. Cells were starved for 24 hours in RPMI with 0.1% sera before receptor tyrosine kinase (RTK) signalling experiments. Cells were treated with 2ng or 40ng/ml recombinant EGF or HGF, respectively, at given time points and lysates analysed by Western blotting and quantified by densitometry.​
Results: Our analysis of TCGA oesophageal adenocarcinoma data set showed expression of Itch mRNA was upregulated in 11% of patient samples (n=186). Itch knockdown in OE33 cells affected ERK1/2 and Akt signalling downstream of the EGFR receptor, and not the Met receptor. Cell signalling assays demonstrated a enhanced pERK (p=0.0478, n=4) and pAkt (p=0.0191, n=4) compared to CTRL Sh cells. Itch knockdown reduced proliferation when grown in 10% sera (p=0.0458, n=4), however under restricted mitogen conditions, Itch depletion enhanced proliferation (p=0.0155, n=4) and viability (p=0.009, n=4) compared to control. Itch was shown to bind to Intersectin-1 (Itsn1) in GST pull-down assays, a protein involved in clathrin-mediated endocytosis. Expression levels of Intersectin-1 have been shown to influence RTK internalisation, thus implicating a novel Itch-mediated regulatory mechanism for membrane trafficking.​
Conclusion: Our study highlights the importance of the regulation of receptor signalling and membrane trafficking in oesophageal cancer to further understand their roles in cell survival, invasion and metastasis. ​
Original languageEnglish
Title of host publicationPharmacology 2017: British Pharmacological Society: Proceedings Annual Meeting
Publication statusPublished - 2018
EventPharmacology 2017 - QEII, London, United Kingdom
Duration: 11 Dec 201713 Dec 2017

Conference

ConferencePharmacology 2017
CountryUnited Kingdom
CityLondon
Period11/12/201713/12/2017

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    Seeley, A., Cobbe, N., Turkington, R., Longley, D., & Evergren, E. (2018). Conference Abstract: Itch e3 ubiquitin ligase influences downstream receptor tyrosine kinase signalling in the oe33 cell line. In Pharmacology 2017: British Pharmacological Society: Proceedings Annual Meeting